When testing on n = 692 healthy people, we found a median (across participant) mean absolute error (within participant) of 9.5 years. Performance was more accurate (MAE around 7 years) in the prefrontal cortex and periventricular areas. We also introduce a new voxelwise method to reduce the age-bias when predicting local brain-age “gaps.” To validate local brain-age predictions, we tested the model in people with mild cognitive impairment or dementia using data from OASIS3 (n = 267). Different local brain-age patterns were evident between healthy controls and people with mild cognitive impairment or dementia, particularly in subcortical regions such as the accumbens, putamen, pallidum, hippocampus, and amygdala. Comparing groups based on mean local brain-age over regions-of-interest resulted in large effects sizes, with Cohen's d values >1.5, for example when comparing people with stable and progressive mild cognitive impairment. Our local brain-age framework has the potential to provide spatial information leading to a more mechanistic understanding of individual differences in patterns of brain ageing in health and disease.We propose a new framework for estimating neuroimaging-derived “brain-age” at a local level within the brain, using deep learning. The local approach, contrary to existing global methods, provides spatial information on anatomical patterns of brain ageing. We trained a U-Net model using brain MRI scans from  Brain ageing is associated with cognitive decline and an increased risk of neurodegenerative disease, though these effects vary greatly between individuals. Brain atrophy, often measured using structural MRI, is commonly seen in many neurological diseases . This approach of using a separate linear regression model for each voxel is limited as it does not incorporate contextual information from neighbouring voxels, and is insensitive to non-linear relationships. Other studies have provided local or regional information by training separate models per region e.g., Kaufmann et al., Limited prior work on local predictions of brain-age are available. Of note, is the early work of Cherubini et al. , who use3 3D block, reporting MAE values between 2.16 and 4.19 depending on block origin. While these approaches are promising, the relatively large size of the patch limits spatial resolution which results in less insightful inference in clinical settings. For example, semantic dementia is associated with a relatively localised spatial pattern of atrophy, often the left anterior and middle temporal lobe  from participants aged 18–88 years, with a mean age of 54.29 ± 18.59 and a male/female ratio of 322/330. More information can be found at https://www.cam-can.org/.This dataset is part of larger project which is trying to use epidemiological, behavioural and neuroimaging data to understand how individuals can best retain cognitive abilities into old age. All participants were scanned with 3 T Siemens TIM Trio scanner with a 32-channel head coil. The dataset consists of n = 494; age range = 19–80 years; mean age 45.18±17.44; male/female ratio of 187/307) undergoing a multi-modal  neuroimaging. Only T1-weighted 3D MPRAGE  were used here. The goals of the SALD are to give researchers the opportunity to map the structural and functional changes the human brain undergoes throughout adulthood and to replicate previous findings. More information can be found at http://fcon_1000.projects.nitrc.org/indi/retro/sald.html.This comprises a large cross-sectional sample  with a 3D T1-weighted MPRAGE sequence . More information can be found at http://fcon_1000.projects.nitrc.org/indi/retro/wayne_11.html.The Wayne State longitudinal data set for the Brain Aging in Detroit Longitudinal Study, comprises 200 healthy individuals, with Here we used data from the Imperial College London project, STudy Of Reliability of MRI (STORM). The study comprises of 20 participants with a male-female ratio of 12/8, with a mean age at the first scan undertaken of 34.05 ± 8.71. The participants were scanned for the second time at an average distance of 28.35 ± 1.09 days. All participants were free from any neurological or psychiatric disorders. MPRAGE were acquired using a Siemens Verio 3T scanner .This study included 11 participants scanned in two different centres, mean age at first scan of 30.88 ± 6.16 and with a male/female ration of 7/4. The two scanning sites were at Imperial College London, where a Siemens Verio 3T scanner was used to acquire MPRAGE , whereas a Philips Ingenia 3T scanner was used at the Academic Medical Center Amsterdam to acquire sagittal Turbo Field Echo . The mean interval between scans was 68.17 ± 92.23 days.n = 609 cognitively normal adults and n=489 individuals at with MCI or dementia ranging in age from 42 to 95 years. Using Clinical Dementia Rating scale (CDR) scores, we classified participants as healthy control (HC), stable MCI, progressive MCI or AD, as detailed in https://www.oasis-brains.org/.This is a retrospective compilation of data for >1,000 participants that were collected across several ongoing projects through the WUSTL Knight ADRC over the course of 30 years. Participants include https://aibl.csiro.au/). The dataset contained n = 198 participants with Clinical Dementia Rating scale scores, detailed in This study is a study to discover which biomarkers, cognitive characteristics, and health and lifestyle factors determine subsequent development of symptomatic Alzheimer's Disease (AD) (https://www.fil.ion.ucl.ac.uk/spm/software/spm12/). This entailed tissue segmentation into grey matter (GM) and white matter (WM), followed by a non-linear registration procedure using the DARTEL algorithm  software package  is used to test the reproducibility of a certain quantitative measurement made by a specified number of observers which rate the same participant. The original formula is given as follows:where Here, we used ICC as defined by Shrout and Fleiss . The intIn this subsection, we summarise the design of our experiments and which datasets are used in each step.BAHC, CamCan, Dallas and SALD were used as training and validation sets (standard 80/20 split) for training the local brain-age U-net model.All Wayne State participants and healthy participants from OASIS3 and AIBL were used at testing time. We calculated mean absolute error (MAE) values globally (by averaging across voxel-level brain-predicted age for each participant) and at voxel level, alongside the Pearson's correlation coefficient between chronological age brain-predicted age at both global and voxel level.Within-scanner reliability and Scanner calibration datasets were used as test sets to compute voxel-level ICC values to assess the reliability of local brain-age when the same participant is scanned in two different scanners, respectively one the same scanner with short time interval.3) for each ROI, alongside ROI-level brain-PAD values which were computed by first averaging voxel-level “brain-predicted age” inside an ROI, then subtracting the participant's chronological age. We calculated a Pearson's correlation coefficient for each ROI.Using subcortical and cortical ROIs from the Harvard-Oxford structural brain atlas, we obtained brain tissues volumes , again computing Cohen's d effect sizes. For all experiments in this part we have selected subjects above 60 years old from the healthy controls so as to have a similar chronological age distribution in relation to the groups with varying degrees of cognitive impairment.OASIS3 was used at testing time to assess the sensitivity of local brain-age to differences in brain structure between groups. For each participant we computed an mean across voxels global brain-PAD (adjusted for age bias using method described in section 2.6), which we use then to perform Welch's A visual overview of the study design is portrayed in 3 voxels. The convolutional layers in our network used an isotropic 3x3x3 filter, convolved over the input image after which element-wise multiplication with the filter weights and subsequent summation was performed at each location. Subsequently, to allow for non-linear modelling, we passed the obtained values through an “activation function”; we used leaky rectified linear units with alpha = 0.2, more precisely max+min, thus allowing a small, non-zero gradient when the unit is not active.We used a fully convolutional neural network (CNN) inspired by the U-Net architecture introduced in Ronneberger et al. . Our netThe convolution operation is also controlled by its stride, which is how many pixels/voxels are skipped after every element-wise weight multiplication and summation. We set the stride equal to 1.Downsampling increases the effective field of view or “receptive field” of layers higher in the hierarchy. For the downsampling part of the U-Net we used at each scale two consecutive 3D 3 × 3 × 3 filter kernels with an initial number of channels = 64, which get multiplied by 2 as we progress down the downsampling path. For downsampling we used 2 × 2 × 2 average pooling.3 voxels blocks.For the upsampling part of the U-Net we inverted the downsampling architecture, with the downsampling layers being replaced by 2 × 2 × 2 upsampling layers. At each convolution we used a squeeze-and-excite unit. Squeeze & Excite networks were introduced in Hu et al.  and can Besides the voxel-level mean absolute error cost function on the output layer we introduced two additional cost functions at the two other scales of the architecture. We applied global average pooling followed by a dense layer to predict brain-age at block-level. The loss function can be expressed as follows:1 and α2 are progressively decreased so that the gradients will exclusively flow from the voxel-level predictions after 50,000 training iterations. We used Adam  or brain-age “delta.” A so-called “regression dilution” has been commonly observed in brain-age prediction algorithms, caused by noise in the neuroimaging features leading to a greater under- or over-estimate of age, the further away a sample is from the training set mean age. In other words, this effect results in the systematic under-estimation of brain-predicted age for older participants and over-estimation for younger participants, which increases as model performance decreases.Broadly speaking, two approaches to account for this effect have been reported:Age representing chronological age.where Δ is the brain-age delta of a group of participants from an external dataset that is used specifically for adjusting the bias. α and β are the parameters of a linear regression with the covariate Then, to obtain the bias-adjusted age we have the following equation:Another approach involves using the brain-predicted age in the linear regression, more specifically:where ỹ denotes the brain-predicted age. de Lange and Cole  showed tn = 200) of participants randomly selected from the healthy participant datasets , who were not included in the training or validation set. We obtained testing time predictions for these participants and calculated their voxel-level brain-age delta Δi, v, where i indicates the i-th participant and v the v-th voxel. We then binned these participants based on their chronological age . Then, for each bin b we calculated the average voxel-level brain-age delta for that respective bin, which we denote as Δb,v. This value will represents the average brain-age delta for that voxel given the chronological age interval. Subsequently, to de-bias the voxel-level brain-age delta for a new participant , Δj,v we used the following formula:Here, we used a separate small batch , AIBL (n = 83) and Wayne State (n = 200) datasets. When looking at voxel-level MAE (unadjusted) values across the brain mask (r = 0.47), for Wayne State 8.09±6.08 years , respectively for OASIS3 we get an average of 8.08±6.40 years . We tested the local brain-age model on healthy participants combined from the OASIS3  and Scanner calibration (between-scanner) datasets, ICC was calculated per voxel. Test-retest reliability was very high with the vast majority of voxels having ICC <0.90  . This inWe examined patterns of local and global brain-age in people with MCI and dementia patients using cross-sectional data from OASIS3. Firstly, we investigated if the global-level  brain-predicted age (adjusted) corresponds to previously reported differences from models that directly predict global brain age. We averaged voxel-level brain-age (adjusted) across voxels per individual to generate an adjusted global-level brain age and then calculate global-level brain-PAD. Global-level brain-PAD (adjusted) mean (± standard deviation) values were: −0.65 ± 7.46 (median = 0.95) years for healthy controls, 3.07 ±4.29 (median = 2.83) years for stable MCI (sMCI), 5.77 ± 5.41 (median = 4.94) years for progressive MCI (pMCI) and 4.34 ± 6.78 (median = 4.63) years for AD patients.t-tests, finding significant differences between cognitively impaired groups and healthy controls in all cases . Comparisons between stable and progressive MCI patients and with AD patients were not significant: sMCI-pMCI p = 0.161, t = −1.44, df = 26.44, Cohen's d = −0.54, AD-sMCI t = 0.83, p = 0.414, df = 20.64, Cohen's d = 0.19, AD-pMCI t = −0.90, p = 0.3714, df = 28.51, Cohen's d = −0.21.We then assessed the significance of group differences using global-level brain-PAD values by performing independent two-sample Welch's t-test. Likewise, differences between participants MCI or dementia and healthy controls were significant . In contrast to the global-level results .Next, we examined local brain-PAD, summarising across all voxels within group. The mean voxel-level brain-PAD (adjusted) values were: healthy controls = −0.39±0.85 (median = −0.44) years, sMCI = 3.07±1.67 (median = 3.266) years, pMCI = 5.45±1.74 (median = 5.663) years for pMCI, AD patients = 4.01±1.71 (median = 4.229) years . We thend scores both for separating AD patients from healthy controls and stable from progressive MCI  is below what has been reported with purely global  including the hippocampus, and the amygdala, entorhinal, and parahippocampal cortices  and amygdala (d = 0.45), when comparing stable and progressive MCI patients. Here, our local brain-age framework resulted in d = 2.18 for the hippocampus and d = 1.5 for the bilateral amygdala in the same context. This suggests that use of the brain-age paradigm to capture local age-related changes, relative to a healthy ageing model, could increase statistical power in experimental research and clinical trials, relative to conventional volumetric imaging biomarkers. Potentially, the ROI-based brain-PAD values could even be used in a classification framework to distinguish between people with stable or progressive MCI.As biomarker of brain health, brain-age models may have clinical utility, either prognostically or in the context of clinical trials of neuroprotective treatments. While previous studies have reported standardised effect sizes from global brain-age, we used atlas ROIs to summarise regional values of local brain-PAD and generated Cohen's n et al.  reportedr et al.  generateOut proposed U-Net local brain-age framework has some strengths and weaknesses. Our model was assessed on a large multi-site testing set with a flat distribution of chronological age across the adult lifespan  was calculated starting from the first convolutional layer where the FOV is 33 voxels, which gets increased by 23 voxels per convolutional operation in the downstream part of the U-Net. The average pooling layers increase the FOV by 13 voxels, since their stride is set to 1, while the upsampling layers do not increase the FOV as they merely repeat existing information. Lastly, the first convolutional layer in the upstream layers only adds 13 voxels  whereas the second adds 33 voxels . While this means that our resolution is not necessarily at the voxel-level, 233 voxels is still substantially higher resolution compared to existing models in literature. In the 3D block approach of Bintsi et al. , in order to encourage the network to emphasise the context encoded in its lower layers. As the individual voxel location we are aiming to obtain a prediction for is not necessarily related to the imposed ground truth output, the U-Net architecture is biased toward using the context information. Hence, in the worst case scenario where no voxel-level relationship is learned, the true resolution of our voxel-level predictions is actually blocks of 23i et al. , blocks One drawback of this study is the categorisation of subjects into sMCI vs. pMCI vs. AD. We attempted to closely follow the ADNI clinical procedure, respectively to classify a subject as AD if they had a mini-mental state examination (MMSE) score between 24 and 26, CDR of 0.5 or 1.0. Subjects with an MMSE score between 24 and 30, CDR of 0.5 alongside memory complains, objective memory loss measured by education adjusted scores and absence of significant levels of impairment in other cognitive domains are classified as MCI. Unfortunately, for OASIS3 we only had access to CDR scores, hence there is the possibility of misclassifications occurring in our data curation.de facto method to quantitatively assess differences between groups at voxel-level , genotype or polygenic risk score, cognitive measures . Such an approach could be used as an alternative to techniques like VBM, to provide mechanistic insights into the relationship between local brain regions and individual deviations from healthy/normal levels of a given outcome measure. While VBM is the One potential direction to take local brain-age further is in disease subtyping. Local brain-PAD maps could be used as input to clustering algorithms with the goal of identifying subgroups of patients that have spatially similar patterns of brain ageing. The putative subgroups may undergoing distinct pathological processes that effect different regions of the brain and may have different trajectories of disease progression or may respond differently to treatments. Such approaches have been applied to volumetric brain maps before  under the European Union's Horizon 2020 research and innovation programme . DS was supported by the NIHR Biomedical Research Centre at Imperial College Healthcare NHS Trust and the UK Dementia Research Institute (DRI) Care Research and Technology Centre. JC acknowledges funding from UKRI/MRC Innovation Fellowship (MR/R024790/2).BG has received grants from European Commission and UK Research and Innovation Engineering and Physical Sciences Research Council, during the conduct of this study and is Scientific Advisor for Kheiron Medical Technologies, Advisor and Scientific Lead of the HeartFlow-Imperial Research Team, and Visiting Researcher at Microsoft Research. JC is a shareholder in and Scientific Advisor to BrainKey and Claritas HealthTech, both medical image analysis software companies. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The results showed that the structure of CUR-Cochs was a spiral cylinder with an average particle size of 463.8 nm and zeta potential of −15.47 mV. The encapsulation efficiency was the highest (83.66 ± 0.8)% with 1:50 CUR-to-lipid mass ratio. In vitro results showed that cochleates had negligible cytotoxicity and owned antioxidant capacity, which provided the possibility for their applications in food and medicine. In general, the method herein might be a promising method to encapsulate CUR for further use as a bioactive agent in functional foods.Curcumin (CUR), a polyphenolic substance extracted from plants, has extensive pharmacological activities. However, CUR is difficult to be absorbed in the body due to its poor stability and low solubility. Studies have found that cochleates can be used as a new delivery system to encapsulate bioactive agents for the purpose of improving its stability and bioavailability. In this study, thin-film dispersion and trapping methods were used to prepare curcumin-loaded cochleates (CUR-Cochs). Then CUR-Cochs were characterized and the encapsulation efficiency was determined by HPLC. In addition, the freeze-drying process of CUR-Cochs was studied and related characterization was performed. CCK-8 assay was used to detect the cytotoxicity of cochleates carrier. Additionally, H CUR, an orange-yellow crystalline powder, is a polyphenolic substance extracted from curcuma plants ,2. It is2+, Zn2+) by electrostatic action. Negatively charged phospholipids are often used [It is reported that many delivery systems could be used to encapsulate CUR, such as micelles, emulsion, liposome, and so on ,18, lipiten used . In geneten used ,24  was purchased from Sinopharm Chemical Reagent Co., Ltd. . Reactive oxygen species (ROS) assay kit was purchased from Beijing Applygen Technology Co., Ltd. . Ultra-pure water was obtained by MILLI-Q* ultra-pure water system.PhosphatidylSerine (PS) was purchased from Chemi Nutra Inc. . Phosphatidylcholine (PC) was purchased from Shanghai Macklin Biochemical Co., Ltd. Curcumin, citric acid, α-D-Lactose monohydrate, trehalose, and mannitol were purchased from Shanghai yuanye Bio-Technology Co., Ltd. . Sodium dihydrogen phosphate and dibasic sodium phosphate were purchased from Sinopharm Chemical Reagent Co., Ltd. . Artificial saliva , simulated gastric fluid (CZ0212), and artificial intestinal juice (CZ0200) were purchased from Beijing Leagene Biotechnology Co., Ltd. . Cell Counting Kit-8 (CCK-8), Dulbecco’s modified Eagle medium (DMEM) and fetal bovine serum (FBS) were obtained from Dalian Meilun Biotechnology Co., Ltd. . Hydrogen peroxide  with 6000 rpm and at 4 °C for 10 min. Finally, transparent and uniform dispersion of CUR-Lipos were obtained by extrusion through a polycarbonate membrane with 0.45 µm pore size. CUR-Cochs were formed by trapping method, which was formed by adding CaCl2 solution (50 mM) slowly (10 µL at a time) to CUR-Lipos with constant low-speed stirring above Tm of the lipid.In this work, thin-film hydration method was used to prepare CUR-Lipos. PS and CUR were dissolved in dichloromethane in a 250 mL eggplant-shaped flask, which was connected to SY-2000* rotary evaporator  to evaporate the solvents at 25 °C with a speed of 60 rpm for 20 min to form a uniform film. Then, the flask was placed in a vacuum drying dish for more than 2 h and blew nitrogen for 5 min to remove residual organic solvents. The film was hydrated with 45 mL ultra-pure water by magnetic stirrer for 1 h above the Tw/v) respectively) were selected as freeze-dried protective agents. Firstly, prepared sam ples were put in a −80 °C refrigerator to pre-freeze for 24 h. After that, samples were put into a Scient-18N* vacuum freeze dryer  for 24 h at −55 °C and a vacuum pressure under 100 Pa to obtain CUR-Cochs freeze-dried powder.The freeze-drying technology was used to prepare freeze-dried CUR-Cochs to increase its storage stability. 4 mL samples were placed in a 10 mL vial respectively, and then different freeze-drying protective agents were added to ensure that the physical and chemical properties of samples remained unchanged before and after freeze-drying. Lactose, mannitol, trehalose, and glucose ; Mobile phase: methanol:1% citric acid ; Column temperature: 30 °C; Flow rate: 1 mL/min; Detector: UV detector; Detection wavelength: 424 nm (CUR); Injection volume: 10 µL.CUR-Cochs with different CUR-to-lipid mass ratios  were prepared, and the encapsulation efficiency (EE) was determined to optimize the best condition. The HPLC was used to determine the content of CUR. The HPLC condition could be set up as below: HPLC Column: Welchrome is the concentration of CUR encapsulated in CUR-Cochs, and Wf is the concentration of total CUR.The determination of total CUR in CUR-Cochs was carried out as follows: Firstly, 200 μL dichloromethane was added in 0.5 mL CUR-Cochs solution and the mixed solution was sonicated for 5 min. Then the demulsified solution was adjusted to 5 mL with methanol followed by extrusion through the 0.22 μm organic microporous membrane and placed in Waters e2695 HPLC  to calculate the total CUR content. Determination of CUR encapsulated in CUR-Cochs: 0.5 mL CUR-Cochs solution was centrifuged at 6000 rpm for 5 min to obtain the precipitate, which was mixed with 200 μL dichloromethane to ultrasonic for 5 min. Then the following steps were the same as above and the final results were obtained by HPLC. EE was calculated as follows in Equation (1). W2 solution was taken in it under the condition of magnetic stirring and above Tm. Additionally, the average particle size and the zeta potential of different samples were detected by the Malvern Nano ZetaSizer ZS . The experimental temperature was set to 25 °C. Then the equilibrium time of samples was set to 120 s and the data obtained each time was the average value of 15 times by the instrument. Each sample was measured three times.Firstly, 1 mL CUR-Lipos was added to a 2 mL centrifuge tube respectively, and then different volumes of 50 mM CaClTEM can be used to observe the morphology of samples. Firstly, a droplet of sample was placed on a carbon-coated copper grid for 4 min. The copper grid was put on a lint-free filter paper to absorb the excess liquid and air dried overnight. At last, the dry copper grid was observed using a JEM-1400 TEM.DSC was used to determine the thermal behavior of the CUR-Cochs. Raw materials of PS, CUR and mannitol and CUR-Cochs freeze-dried powder were respectively weighed in a certain amount (5–15 mg) and placed in an aluminum crucible with a thin layer on the bottom. The aluminum lid was pressed tightly and pierced with a small hole. In addition, the crucible containing the sample was placed inside placed in the calorimeter and compared with the empty one. In case of the heating rate of 10 °C/min, DSC scan results were performed. Finally, the thermal properties of samples were analyzed according to their heat flow curves.Phosphate buffered saline (PBS) solutions with different pH values  were prepared by sodium dihydrogen phosphate and dibasic sodium phosphate. Then CUR-Lipos and CUR-Cochs were added to PBS solutions respectively, and incubated in a dark water bath at 37 °C. After 2 h, samples were dissolved with methanol to detect the content of CUR by HPLC to evaluate whether cochleates could improve the stability of CUR. The CUR content of original solution was taken as 100% to calculate the CUR content changes at different pH.The effect of heating for different temperatures on the stability of CUR-Cochs was examined comparing with the CUR-Lipos group. 4 mL CUR-Lipos and CUR-Cochs solutions were added into 5 mL vials in five different groups, and then heated in a water bath at 37, 40, 60, 80, and 100 °C respectively for 2 h. Among them, the freshly prepared CUR-Cochs solution stored at 4 °C was used as the control group. After the treatment, methanol was added to demulsify in each vial, and then the content of CUR was measured by HPLC. The CUR retention rate was determined to investigate the thermal stability of CUR-Cochs. The CUR content of original solution was taken as 100% to calculate the CUR content changes at different temperature.In this study, the bioaccessibility of CUR in CUR-Cochs was determined by an in vitro digestion model, which was a simulated gastrointestinal tract consisting of mouth, stomach, and small intestine phase ,26.Mouth phase—The prepared CUR-Cochs suspension was poured into a conical flask, then was preheated in a constant temperature water bath at 37 °C. The prepared artificial saliva fluid was poured into the conical flask and mixed with CUR-Cochs at a mass ratio of 1:1, and the pH of the mixed solution was 6.8. Subsequently, the resulting mixed solution was placed in a shaker and shaken with the speed of 90 rpm for 10 min at 37 °C to simulate oral conditions.Stomach phase—Firstly, simulated gastric juice containing pepsin was prepared and the pH value was 2.5. Then the simulated gastric juice was added to the sample produced by the mouth phase at a mass ratio of 1:1, and the mixture continued to be cultured in a shaker for 2 h at a speed of 100 rpm in 37 °C.Small intestine phase—Samples in the simulated gastric phase were adjusted to pH 7.0 with 1 M NaOH solution, and then the prepared small intestinal fluid was added. The pH of the resulting mixture was kept constant at pH 7.0 by adding 50 mM NaOH solution. The samples were kept for 2 h at 37 °C.After digestion, CUR dissolved in the mixed micellar phase was generally considered to be available for assimilation, since certain mixed micelles were small enough to be transported across the mucus layer to epithelial cells . TherefoInitial is the initial concentration of CUR in cochleates before digestion reaction, CDigesta is the concentration of CUR in overall raw digesta, CMicelles is the concentration of CUR dissolved in micelles fraction.Herein, C4 cells/mL (200 µL/well). After incubated at 37 °C in 5% CO2 incubator for 24 h, 0.4, 0.8, 1.2, 1.6, and 2 M Blank-Lipos and Blank-Cochs solutions were added to each well, and then incubated for 24 h. Before measuring the cytotoxicity, the 96-well plate was placed under an inverted microscope to observe the cell morphology. The culture medium was replaced with 10% CCK-8 solution in 200 μL medium and co-incubated with cells for another 2 h. The absorbance at 450 nm was recorded using a microplate reader, which represented the cell viability. Cell viability (%) was calculated by using Equation (4). Among them, As represented the absorbance of experimental wells, Ac represented absorbance of control wells and Ab represented the absorbance of blank wells.Cell Counting Kit-8 (CCK-8) assay was performed to detect cytotoxicity. Firstly, cells were added into 96-well plates at the density of 1 × 102 at 37 °C for 24 h. Then the culture medium was discarded, washed with PBS 2 or 3 times, and 2 mL culture medium containing the sample was added separately. The normal control group (only the medium without H2O2 solution and samples), model group (the medium without samples directly treated with H2O2 solution) and test groups were set up. Among them, test groups were also added 2 mL culture medium including CUR-Cochs solutions which contained 0.75, 1.5 and 3 µM CUR, 3 µM CUR solution (dissolved in DMSO), and Blank-Cochs solution. After samples were processed for 12 h, 600 µM H2O2 solution was added to each well for 8 h. Then the cells were washed with PBS and collected in centrifuge tubes, and then 1 mL culture medium containing 10 µM 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) was added respectively. After incubating in the incubator for 30 min in the dark, the residual probes were washed with PBS, cells were collected and resuspended in PBS, and then the ROS value was measured by flow cytometry .The antioxidant injured model was set up to determine the protection effects of the CUR-Cochs as described . First op-value of <0.05 was considered statistically significant. All pictures were made with Origin 8.5 and AI 2018 software.All experiments were performed in triplicate and results are expressed as mean ± SD. The data was analyzed with SAS 9.2 software. The single factor method was used to analyze the significance of the difference, and the least significant difference was compared in pairs. A 2, as a cationic linking agent, can bind to the negatively charged head group of CUR-Lipos to open the original structure of the liposome and form a new cochleate structure. Therefore, the content of CaCl2 is critical to the formation of cochleates  was dropped into CUR-Lipos solution, the structure of CUR-Cochs could be found through an optical microscope   C. As shoLactose, mannitol, trehalose, and glucose  were selected as freeze-drying protective agents. It was found that the freeze-dried effect of the mannitol group was the best which observed from the appearance of freeze-dried samples, and the results were shown in p < 0.05). However, the particle size distributions of samples with 10% mannitol or 15% mannitol were not uniform, and there was significant difference in zeta potential between 15% mannitol CUR-Cochs and CUR-Cochs original solution. According to the experiment, 5% mannitol was selected as the freeze-drying protective agent. The difference in zeta potential values after addition of mannitol was caused by the physical characteristic changes of the CUR-Coch. With adding 5%, 10%, and 15% concentration of the mannitol as freeze-drying protect agents, the ability of re-dispersion after dissolve in water of the CUR-Coch powder was different. The optimized 5% mannitol group shown the no significant difference of in size and zeta potential (−23.3 mV) with the original one (−20.8 mV). For the 10% and 15% mannitol groups, the particle sizes were increased to micro sizes and the aggregation can be observed. The forming of larger particle sizes as well as the aggregation may lead to the shielding of more negatively surface charges and probably the reason to cause the gradually reduced zeta potential (−17.2 mV in 10% group and −14.3 mV in 15% group).From As shown in The chemical properties of CUR itself are unstable due to many pairs of carbon-carbon double bond structures in its molecular structure. Therefore, CUR is sensitive to environmental conditions such as high temperature, light, extreme pH, and so on. In this work, PS was used as a material to prepare cochleates as a bio-delivery system to embed CUR and improve the stability. This work discussed that whether cochleates encapsulation could improve the stability of CUR comparing with liposome.p < 0.05). In addition, it could find that the stability of samples was low at pH 9. The results showed that the pH stability of CUR-Cochs was significantly improved compared to CUR-Lipos (p < 0.05). Under neutral or weakly acidic conditions, samples had better stability, while CUR in samples were extremely unstable under alkaline conditions.It has been demonstrated by many studies that the decomposition of CUR depended on different pH values, and CUR was relatively stable under acidic to neutral conditions but degraded faster in alkaline environment . Under wp < 0.05). This showed that high temperature still had a certain effect on the stability of CUR-Cochs, but was more stable than CUR-Lipos. In general, the structure of cochleates was stable, which could well protect the CUR in the phospholipid bilayer and significantly improved the thermal stability of free CUR [CUR is a heat-sensitive biologically active substance, so it was essential to test the thermal stability of CUR-Cochs. Cochleates solutions may face different temperatures during processing, transportation, and storage during the application of the food industry. In this study, CUR retention was used to reflect the thermal stability of the samples in different temperature. As shown in free CUR . At highBioavailability refers to the speed and extent to which the active ingredient enters the human circulation after being absorbed. CUR has many biological and pharmacological activities that may benefit human health . HoweverThe cytotoxicity of Blank-Lipos and Blank-Cochs to L929 cells was detected by the CCK-8 assay. As shown in 2O2 injury, the level of ROS increased to approximately 390% of that in control group. Comparing with the H2O2 group, there was no obvious change in the Blank-Cochs group. Both CUR and CUR-Cochs groups could reduce the increase of cellular ROS induced by H2O2, and the CUR-Cochs treatment group showed a good dose-dependence. At the concentration of CUR was 3 μM, the ROS level of the CUR and CUR-Cochs groups were 131% and 150% of that in control group respectively. The above experimental results showed that both CUR and CUR-Cochs had antioxidant effects, thereby protecting cells from oxidative damage. Among them, the protective effect of CUR was slightly better than that of CUR-Cochs, because CUR in this work was dissolved by DMSO and had better dispersibility. In fact, CUR is insoluble in water without adding DMSO solvent when applied. Future research could mostly focus on how to evaluate the potential toxicity of the CUR-Coch nanoparticle as in vivo delivery system. Additionally, considerable effort should be involved to investigate the mechanism of the CUR-Coch for their further application.ROS plays a dual role in the biological system. Low levels of ROS can participate in the regulation of cell survival signals, and high levels of ROS can damage cell structures and cause oxidative damage or even apoptosis, which is often the main cause of cellular oxidative damage ,39. In tp < 0.05). In addition, the results indicated that CUR-Cochs could improve the bioaccessbility of CUR. Moreover, the cytotoxicity experiments showed that PS as the carrier material was non-toxic by CCK-8 assay. The study of intracellular ROS assay has shown that CUR-Cochs could maintain the ability of anti-oxidation of the CUR and reduce the ROS level in the oxidative damage model with a dose-dependent effect. In general, the prepared CUR-Cochs exhibit the ability of antioxidant with relative low cytotoxicity, which could be potentially to be developed as functional foods or as foods additives. The mechanism of the CUR-Cochs for their antioxidant effect should be further investigated for better application. Additionally, more experiments should be performed to optimize the suitable condition from batch to scale up for their applications. Cochleates technology could also provide a new idea and method for improving the performance of hydrophobic biologically active substances.In this work, PS was used as the carrier material, and the film dispersion method together with the ultrasonic hydration method was used to prepare CUR-Lipos, and then the trapping method was used to formulate CUR-Cochs. The encapsulate rate of CUR in cochleates was about 83.66% with 1:50 CUR-to-lipid mass ratio. The prepared CUR-Cochs were characterized with an average particle size of 463.8 nm and zeta potential of −15.47 mV. In addition, the freeze-drying process of CUR-Cochs was also studied. The results showed that the freeze-dried powder formed with 5% mannitol as the freeze-dried protective agent had the best effect. Comparing with CUR-Lipos, the results revealed that the stability of CUR loaded in CUR-Cochs could significantly improve in different pH and temperature (


Fetal alcohol spectrum disorders (FASDs) are one of the most common consequences of ethanol exposure during pregnancy. In adulthood, these disorders can be manifested by learning and memory deficits and depressive-like behavior. Ethanol-induced oxidative stress may be one of the factors that induces FASD development. The mammalian target of the Rapamycin (mTOR) signaling pathway that acts via two distinct multiprotein complexes, mTORC1 and mTORC2, can affect oxidative stress. We investigated whether mTOR-dependent or mTOR-independent mechanisms are engaged in this phenomenon. Thus, Rapamycin—a selective inhibitor of mTORC1, Torin-2—a non-selective mTORC1/mTORC2 inhibitor, and FK-506—a drug that impacts oxidative stress in an mTOR-independent manner were used. Behavioral tests were performed in adult (PND60-65) rats using a passive avoidance (PA) task (aversive learning and memory) and forced swimming test (FST) (depressive-like behaviors). In addition, the biochemical parameters of oxidative stress, such as lipid peroxidation (LPO), as well as apurinic/apyrimidinic (AP)-sites were determined in the hippocampus and prefrontal cortex in adult (PND65) rats. The rat FASD model was induced by intragastric ethanol (5 g/kg/day) administration at postnatal day (PND)4–9 . All substances (3 mg/kg) were given 30 min before ethanol. Our results show that neonatal ethanol exposure leads to deficits in context-dependent fear learning and depressive-like behavior in adult rats that were associated with increased oxidative stress parameters in the hippocampus and prefrontal cortex. Because these effects were completely reversed by Rapamycin, an mTORC1 inhibitor, this outcome suggests its usefulness as a preventive therapy in disorders connected with prenatal ethanol exposure. Fetal alcohol spectrum disorder (FASD) is the most severe consequence of prenatal alcohol exposure (PAE) during pregnancy. The most profound effects of PAE are neurobehavioral deficits, including physical and cognitive disabilities ,2. TheseSuch effects may be induced by the influence of ethanol on neurogenesis and on myelination of the central nervous system (CNS) during brain development . A critiThe mechanisms responsible for FASD-induced neurodegeneration are multifaceted and complex. One such mechanism is oxidative stress ,11. The The mTOR serine/threonine kinase is an enzyme that, among other effects, regulates cell growth and protein homeostasis via two distinct complexes, mTORC1 and mTORC2. As research suggests, chronic ethanol exposure activates mTOR and causes impairments of the autophagy ,14. ThisThe present study investigated the hypothesis that development of emotional learning deficits and depressive-like behaviors in adult rats exposed to ethanol during the neonatal period are a function of oxidative stress that, in turn, depends on the mTOR signaling pathway. To evaluate this hypothesis, we used Rapamycin—a selective mTORC1 inhibitor, Torin-2—a non-selective mTORC1/C2 inhibitor, and FK-506—an inhibitor of oxidative stress that acts independently to mTOR. To conduct such study, the male rat pups received these substances as pre-treatment before ethanol  administration during PND4–9 so as to protect their brain cells against oxydative stress development. As adults (PND60), aversive learning and memory processes were tested by means of the inhibitory passive avoidance (PA) task ,20,21,22p < 0.01) and ethanol treatment  = 8.232; p < 0.01) on LI values of fear learning and memory and interaction between these factors  = 7192; p < 0.001). However, post-hoc analysis (Tukey’s test) revealed that only the ethanol-exposed group had significantly decreased LI values in rats in the PA task, compared to the control group (p < 0.001). This amnestic effect of ethanol was significantly reversed by Rapamycin pre-treatment at the dose of 3 mg/kg (p < 0.001), but not by Torin-2 and FK-506 pre-treatment at the dose of 3 mg/kg (p < 0.05) (p > 0.05).In adult male rats, two-way ANOVA indicated a statistically significant effect of pre-treatment with the used substances   = 6.120;  < 0.05) . Rapamycp < 0.001) and the ethanol treated groups  = 28.89; p < 0.001) on depressive-like behavior, as well as significant interactions between these two factors  = 8.352; p < 0.001). Post-hoc analysis (Tukey’s test) revealed that the ethanol-exposed group exerted a statistically significant depressive-like effect (p < 0.001). This was manifested in the reduction of total immobility time, in comparison to the control group. Pre-treatment with only Rapamycin had an anti-depressive effect p < 0.001). Torin-2 and FK-506 did not significantly decreased immobility time (p < 0.001). Rapamycin alone pre-treatment at the dose of 3 mg/kg did not change the outcome of the undertaken experiments (p > 0.05), but Torin-2 and FK-506 at the dose of 3 mg/kg did (p < 0.05)  indicated that there were statistically significant differences between pre-treated groups with the used substances   = 31.22;  < 0.05) .p > 0.05) at the dose of 3 mg/kg and ethanol-treated groups  = 3.921; p > 0.05) with regard to locomotor activity, and no significant interactions between these two factors  = 1.978; p > 0.05)  indicated that there were no statistically significant differences between pre-treated groups with Rapamycin, Torin-2, and FK-506  = 0.6464;  > 0.05) .p > 0.05) and prefrontal cortex  = 2.942; p > 0.05), and also the significant effect of ethanol on LPO concentrations in the hippocampus  = 95.08; p < 0.001) and prefrontal cortex  = 89.68; p < 0.001). Two-way analysis also indicated significant interactions between these two factors in the hippocampus  = 6.202; p < 0.01) and prefrontal cortex  = 6.926; p < 0.01). Two-way analysis of variance (ANOVA) indicated the statistically significant effect of pre-treatment with Rapamycin, Torin-2, and FK-506 at the dose of 3 mg/kg on LPO concentrations in the hippocampus  = 3.880; p < 0.001) compared to the control group. Pre-treatment with Rapamycin alone (p < 0.001) decreased this concentration, but pre-treatment with Torin-2 and FK-506 (p > 0.05) did not. The pre-treatment of Rapamycin alone at the dose 3 mg/kg also did not change the outcome of the undertaken experiments (p > 0.05), but pre-treatment at the dose 3 mg/kg with Torin-2 (p < 0.001) and FK-506 (p < 0.001) did  in LPO concentrations in the hippocampus revealed that the ethanol-exposed group had increased ROS production (001) did A.p < 0.001) compared to the control group. Pre-treatment with Rapamycin alone (p < 0.001) decreased this concentration, but pre-treatment with Torin-2 and FK-506 (p > 0.05) did not. Rapamycin alone pre-treatment at the dose of 3 mg/kg did not change the outcome of the undertaken experiments (p > 0.05), but pre-treatment at the dose of 3 mg/kg with Torin-2 (p < 0.01) and FK-506 (p < 0.0001) did  showed that the ethanol-exposed group had increased ROS production (001) did B.p > 0.001) and prefrontal cortex  = 4.580; p > 0.01) and also the significant effect of ethanol treatment on AP site levels in the hippocampus  = 12.17; p < 0.001) and prefrontal cortex  = 8.353; p < 0.001). Two-way analysis also indicated significant interactions between these two factors in the hippocampus  = 68.55; p < 0.01) and prefrontal cortex  = 38.04; p < 0.01).Two-way analysis of variance (ANOVA) indicated a statistically significant effect of pre-treatment with Rapamycin, Torin-2, and FK-506 on AP site levels in the hippocampus  = 9.930; p < 0.001), compared to the control group. Pre-treatment with Rapamycin (p < 0.001) decreased this AP site level and ROS generation, but pre-treatment with Torin-2 and FK-506 (p > 0.05) did not. Rapamycin alone pre-treatment at the dose of 3 mg/kg did not change the outcome of the undertaken experiments (p > 0.05), but Torin-2 and FK-506 pre-treatment at the dose of 3 mg/kg (p < 0.05) did  in AP site levels in hippocampus showed that the ethanol exposed group had increased damage generated by ROS (.05) did A.p < 0.001), compared to the control group. Pre-treatment with Rapamycin also decreased this AP site level and ROS generation (p < 0.001), but Torin-2 and FK-506 did not (p > 0.05). Moreover, Rapamycin alone pre-treatment at the dose 3 mg/kg did not change the outcome of the undertaken experiments (p > 0.05), but Torin-2 and FK-506 pre-treatment at the dose of 3 mg/kg (p < 0.05) did  of AP site levels in the prefrontal cortex revealed that the ethanol-exposed group had increased damage generated by ROS (.05) did B.The current study demonstrated that the adult male rats exposed to ethanol during the neonatal period (PND4–9) showed behavioral deficits in fear learning and memory (according to the PA task results). Furthermore, our findings indicated that neonatal ethanol exposure induced depressive-like behavior in adult male rats according to the forced swimming test. Biochemical analysis also revealed an increase in LPO and AP sites in the prefrontal cortex and hippocampus of adult rats exposed to ethanol during the neonatal period. These data suggest that ethanol-induced cognition deficits and depressive-like behavior are related to increased oxidative stress in these brain structures during neonatal ethanol exposure. Pre-treatment with various mTOR-dependent or independent inhibitors of oxidative stress before ethanol administration during the neonatal period mitigated learning and memory impairment, prevented the depressive-like behavior, and attenuated the oxidative stress induced by ethanol. The most potent effects were observed after pre-treatment with Rapamycin, a selective mTORC1 inhibitor. Administration with this substance normalized these parameters to the values of the corresponding control groups and effectively prevented the harmful effects of ethanol on the developing brain.Our study shows deficits in fear learning and memory in adult rats that received ethanol during the neonatal period. Thus, our results support the observations of other rat-based  and C57 Our findings indicate that Rapamycin, the mTORC1 selective inhibitor, given before ethanol administration during the neonatal period, reversed these ethanol effects. Furthermore, Torin-2—a non-selective mTORC1/C2 inhibitor—and FK-506—a substance that impacts oxidative stress in a mTOR-independent manner—did not prevent fear learning and memory impairments, although the results were partially lowered by these compounds. This means that Rapamycin pre-treated animals spent more time in the bright compartment and less entered the dark compartment where they had been previously shocked. Furthermore, our study confirms the participation of mTORC1 in this preventive mechanism and indicates that the non-selective mTORC1/C2 inhibitor and other substances without impact on mTOR pathway, do not show such behavior. Thus, our outcome suggests that only mTORC1 is engaged in FASD-induced learning and memory deficits. It should be noted that other factors such as ethanol-induced hyperlocomotion can affect the results in this task. However, increase in locomotor activity is restricted to adolescence because it was not observed in the adult (PND61) rats in our study . This fiVarious psychiatric illnesses, including depression, that are recognizable in young children and in adults ,37 are aAlthough Rapamycin possesses anti-inflammatory attributes, our study was focused on its anti-oxidant properties . Thus, mIn ethanol-induced neurodegeneration affecting the brain, DNA damage induced by oxidative stress is also one of the major factors leading to neuronal dysfunction and cell death. Studies have shown that in brain structures such as the hippocampus and prefrontal cortex, a relationship exists between DNA damage and the presence of cortisol, especially during prolonged exposure to high levels of cortisol . OxidantPrevious studies have shown that oxidative DNA damage is linked to the onset of specific human diseases such as neuronal degeneration ,54,55. OThe exact mechanism of Rapamycin involvement in FASD development and potential protection still remains unknown. Oxidative stress, which is caused by expressive production of ROS, has been proposed as a one of the mechanisms for ethanol-induced neuronal damage and is associated with modulation of behavioral processes underlying the performance of cognition and emotions. To explore this hypothesis, we evaluated the effect of various modulators of mTOR so as to ascertain which pathway is involved in oxidative stress and inhibition, as well as which substance has potential therapeutic value. The present study confirms that FASD may be a result of oxidative stress. In our study, the level of LPO and AP site damage was increased in the hippocampus and prefrontal cortex, two brain regions involved in learning and memory and depression. Moreover, these data support the previous findings that prenatal ethanol exposure is associated with an increase in oxidative stress in the brain regions involved in neurodegeneration . The effA recent study confirmed that oxidative stress in ethanol-treated rats brings about damage to mitochondria , and results in the production of intracellular reactive oxygen . Our datTaken together, our findings suggest that Rapamycin, as a selective inhibitor of mTORC1, can act neuroprotectively in response to ethanol neurotoxicity, and that it alleviates ethanol-mediated oxidative stress and neuronal death. It is possible that the described changes are dependent on autophagy flux, hence, the exact mechanism of such interactions needs further evaluation. Overall, our work reveals that mTORC1 inhibitors play an important role in depressive-like behaviors and learning functions in adult rats that received ethanol during the neonatal period and may be useful as a preventive therapy in disorders connected with prenatal ethanol exposure.Wistar rats  were bred and housed in a vivarium under standard laboratory conditions: a light/dark cycle of 12 h/12 h, consistent temperature of 22 ± 1 °C, controlled humidity of 55 ± 10%, and free access to standard laboratory chow  and water ad libitum. For breeding, one male and one female rat were housed together for one week. Beginning 3 weeks after mating, the females were checked twice a day for parturition. Following parturition, on PND3, male pups were assigned to double experimental groups  8 pups each and retaining equal numbers (see below). All rats  were housed with dams to PND21 then housed with 2–3 littermates through PND59. Our experiments used only male rats. This is because, according to prior research ,67,68, mw/v, Polmos, Poznan, Poland) was given on PND4-9 as a “3rd trimester exposure” model. Pups received ethanol via intragastric intubation (i.g.)  delivered in a milk (Bebilon 1 Pronutra Plus) solution. This dose of ethanol produces significant neurotoxicity during the third trimester equivalent and may lead to neurobehavioral deficits [On PND3, male rat pups were paw-marked for identification and were assigned into 1 of 8 treatment groups that were used in the behavioral and biochemical experiments: 0.9%NaCl + sham intubated, Rapamycin + sham-intubated (3 mg/kg), Torin-2 (3 mg/kg) + sham-intubated, Fk-506 (3 mg/kg) + sham intubated, 0.9%NaCl + ethanol, Rapamycin (3 mg/kg) + ethanol, Torin-2 (3 mg/kg) + ethanol, or FK-506 (3 mg/kg) + ethanol see . Inductideficits . The othdeficits , the Tordeficits . Sham-inThe passive avoidance procedure produces similar results in both mice and rats . The PA The PA procedure was based on the association formed between an aversive stimulus (foot shock of 0.15 mA intensity and 2 s duration) and a specific environmental context (dark compartment). The experiment consisted of three steps: habituation (PND60), training (PND60), and retention testing (PND61). The procedure was as follows.All rats were allowed to habituate in the experimental room for at least 30 min prior to the experiments. Next, in the training session, each animal was gently placed in the light compartment of the apparatus and allowed to freely explore. After 30 s, the guillotine door was opened, and the rat was allowed to enter the dark compartment. When the rat entered the dark chamber, the guillotine door was closed and the electric foot-shock duration was delivered immediately to the animal via the grid floor of the dark room by an insulated stimulator. Next, the animal was removed from the apparatus and placed in its home cage. The latency with which the animal crossed into the dark compartment was recorded (TL1). If the rat failed to enter the dark box within 300 s, it was placed into this dark box, the door was closed, and the electric foot-shock was delivered to the animal. In this case, TL1 value was recorded as 300 s. On PND61, 24 h later, the retention test trial was conducted with exactly the same conditions, but without electric shock. The latency with which the animal crossed into the dark compartment was recorded (TL2) and the time spent in the dark compartment was measured up to 300 s. If the animal did not enter the dark chamber within 300 s, the test was terminated and TL2 was recorded as 300 s.To assess memory-related behaviors, the changes in the PA task were expressed as the difference between training and test latencies and were taken as the latency index (LI). LI was calculated for each animal as the ratio: LI = TL2-TL1/TL1, where: TL1—the time taken to enter the dark compartment during the training and TL2—the time taken to re-enter the dark compartment during the retention . A high The experiment was carried out using the test proposed by  to examiThe FST procedure consisted of a habituation (PND64) and test phase (PND65). The procedure was as follows.During the habituation phase, on the first day of the experiment, each rat was placed individually in a glass cylinder (10-liter capacity) filled with water (23–25 °C) for 15 min in order for it to adapt to the new conditions. After this time, the animals were returned to their home cages. On PND65, 24 h later, the test phase was conducted. The animals were placed individually in the water-filled cylinder again for 5 min and the total immobility time was measured by means of stopwatch. Immobility was assumed when the animal floated passively , performing only enough movement necessary to keep its head above water .2) made of transparent plexiglass and was designed for measuring locomotor activity . The cages were located in a sound-attenuated experimental room with constant lighting (40 W). The procedure was as follows, and was based on a method used in previous studies [The locomotor activity of individual rats was recorded using a photocell apparatus  and was undertaken to exclude the impact of locomotor on the PA and FST results. The apparatus was a square cage  were placed in the square Plexiglas cage. After 10 min of acclimatization, the total horizontal activity (distance traveled in meters) of individual animals was measured (15 min) by two rows of infrared light-sensitivity photocells placed 45 and 100 mm above the floor. To remove odor traces, the apparatus was thoroughly cleaned with water between each individual test run.On the last day of behavioral experiments (PND65), the animals were humanely killed and the prefrontal cortex and hippocampus were isolated from the rats’ brains. During the next step, tissue was homogenized from the frozen brain samples using extraction buffer. All biochemical measurements were conducted from these homogenates. The experimental procedures were performed according to the instructions supplied with each respective kit.In this experiment, lipid hydroperoxide (LPO) testing was undertaken. LPO was based on malondialdehyde and 4-hydroxyalkenals concentration (MDA + 4HAE) . The principle underlying LPO assessment is based on the reaction of a chromogenic reagent R1 (N-methyl-2-phenylindole) with malondialdehyde (MDA) and 4-hydroxyalkenals (4HAE) at 45 °C. Two molecules of R1 react with one molecule of MDA or 4-hydroxyalkenals to form a chromophore with an absorbance maximum at 586 nm. Measuring the concentration of MDA in combination with 4-hydroxyalkenals in methane sulfonic acid was used as an indicator of LPO level.DNA was isolated with a Syngen DNA Mini Kit  according to the manufacturer’s protocol. The concentration and purity of the genomic DNA were measured using a NanoDrop MaestroNano Micro-Volume Spectrophotometer  and adjusted to 100 μg/mL in TE buffer. Oxidative DNA damage was evaluated by measuring the number of basic sites  with a DNA Damage Quantification Kit . Oxidative attacks by ROS on the deoxyribose moiety lead to the release of free bases from DNA, generating strand breaks with various sugar modifications and simple basic sites (AP sites). The aldehyde-reactive probe  reacts specifically with an aldehyde group present on the open ring form of AP sites, making it possible to detect DNA modifications that result in the formation of an aldehyde group. Biotin-avidin-specific connection and horseradish peroxidase were used for colorimetric detection at 650 nm. AP sites were measured in the DNA isolated from the hippocampus and prefrontal cortex of the rats.p value of less than 0.5 was considered statistically significant for all testsThe results were analyzed using the GraphPad Prism version 8.00 for Windows, GraphPad Software . The statistical significance of drug effects and the behavioral and biochemical tests were analyzed by two-way analysis of variance (ANOVA) with repeated measures. This was followed Tukey’s post-hoc test. The results were presented as means ± standard errors of means (SEM) of values. A Collectively, the present data demonstrate that the mTORC1 signaling pathway is responsible for the neurodegenerative effects of alcohol on brain development during the neonatal period. Moreover, these data reveal that such effects are associated with increased oxidative stress parameters in the hippocampus and prefrontal cortex. Our results also suggest that Rapamycin (a selective mTORC1 inhibitor), but not Torin-2 (a non-selective mTORC1/C2 inhibitor) or FK-506 (which has an impact on oxidative stress in a mTOR-independent manner), could have protective effects against the ethanol-induced LPO and AP site levels in the hippocampus and prefrontal cortex. Both of the latter are indicators of oxidative processes. However, a better understanding of the mTORC1 mechanism and oxidative stress are needed to produce effective therapy against neurotoxicity induced by neonatal ethanol exposure.


Some patients with early-stage oral tongue cancer suffer from poor survival. The currently used classification requires further improvement to better predict the prognosis. Immune-related parameters (such as assessment of infiltrating lymphocytes) can be used as a modifier for the classification and that can aid in improving the prognostication. We included 290 cases of early-stage oral tongue cancer in this study. Lymphocytes were scored and divided as low or high and incorporated in the traditional tumor-node-metastasis (TNM) classification to form our proposed TNM-Immune staging system. The TNM-Immune staging system allowed for a significant distinction between T1 and T2. The TNM-Immune staging system showed a powerful ability to identify cases with poor survival. TNM-Immune staging forms a step towards a more personalized classification of early-stage oral tongue cancer.p > 0.05). Our proposed TNM-Immune staging system allowed for significant discrimination in risk between tumors of T1N0M0-Immune vs. T2N0M0-Immune. The latter associated with a worse overall survival with hazard ratio (HR) of 2.87 ; HR of 2.41  for disease-specific survival; and HR of 1.97  for disease-free survival. The TNM-Immune staging system showed a powerful ability to identify cases with worse survival. The immune response is an important player which can be assessed by evaluating TILs, and it can be implemented in the staging criteria of early OTSCC. TNM-Immune staging forms a step towards a more personalized classification of early OTSCC.Although patients with early-stage oral tongue squamous cell carcinoma (OTSCC) show better survival than those with advanced disease, there is still a number of early-stage cases who will suffer from recurrence, cancer-related mortality and worse overall survival. Incorporation of an immune descriptive factor in the staging system can aid in improving risk assessment of early OTSCC. A total of 290 cases of early-stage OTSCC re-classified according to the American Joint Committee on Cancer (AJCC 8) staging were included in this study. Scores of tumor-infiltrating lymphocytes (TILs) were divided as low or high and incorporated in TNM AJCC 8 to form our proposed TNM-Immune system. Using AJCC 8, there were no significant differences in survival between T1 and T2 tumors ( Oral tongue squamous cell carcinoma (OTSCC) is the most common and aggressive SSC of the oral cavity. The most clinically relevant parameter in the classification of OTSCC is the tumor-node-metastasis (TNM) staging system which evaluates tumor size (T), lymph node status (N) and distant metastasis (M) . For earData accumulating on the significance of adaptive immune response, regardless of histologic grade, were reported for different cancers and this approach has shown promising results that can be useful in routine practice ,4. HowevThis study was conducted with the permission of the National Supervisory Authority for Welfare and Health in Finland and the Brazilian Human Research Ethics Committee. A total of 290 cases treated for OTSCC at the five Finnish university hospitals or at the A-C Camargo Cancer Center in São Paulo, Brazil, and re-staged according to the criteria of the TNM AJCC 8 were included in this study. We included only naïve tumors that were treated by surgical resection. Our data did not include cases with immunosuppression or previous chemotherapy. As our analysis considered only early-staged OTSCC, all cases were either T1N0M0 or T2N0M0 according to the criteria of the TNM AJCC 8. Our analysis aimed to compare the prognostic value of the classic TNM AJCC 8 with that of our proposed TNM-Immune staging system in predicting three survival endpoints: overall survival (defined as the time from surgery to death of any cause), disease-specific survival (defined as the time from surgery to death due to OTSCC), and disease-free survival (defined as the time from surgery to recurrence).Stromal TILs were defined as stromal areas occupied by lymphocytes as explained in our previous study . The assOur proposed TNM-Immune classification is designed based on the TNM AJCC 8 and includes the status of preexisting immunity as revealed by evaluation of TILs as follows:T1: Tumor ≤ 2 cm, ≤5 mm depth of invasion, and TILs > 20%.T2: Tumor ≤ 2 cm, DOI > 5 mm and ≤10 mm; or tumor > 2 cm but ≤4 cm, and ≤10 mm DOI. TILs should be ≤20% , otherwise downstaging is necessary.p value of less than 0.05 was considered significant. Age, gender, perineural invasion, tumor grade, and TNM stage were included in the multivariable analysis in addition to our proposed TNM-Immune staging system.Statistical analysis: We used SPSS Statistics software (version 25) for all statistical analyses. Pearson Chi-Square test (two-sided) was used to analyze the correlation between the TNM-Immune staging system and the traditional parameters. The Kaplan–Meier method and log-rank test were used to create survival curves. The univariable and multivariable analyses were conducted using a cox proportional hazard regression. A The demographic and clinicopathologic data of 290 patients are shown in p = 0.037) and perineural invasion (p = 0.013). However, there was no significant association between TNM-Immune stage and tumor grade, patient age or gender (p > 0.05). In univariate analysis of TNM AJCC 8 (p = 0.473) was reported for overall survival, a HR of 1.36  was reported for disease-specific survival, and a HR of 0.69  for disease-free survival. The multivariable analyses summarized in p > 0.05) between cases that were early-stage OTSCC as classified by AJCC 8.The cross-tabulation  showed aM AJCC 8 , a hazarn = 290), the newly proposed TNM-Immune staging system allowed for a significant risk stratification between cases staged as T1N0M0-Immune compared to cases of T2N0M0-Immune. We found that T2N0M0-Immune associated significantly with a worse outcome with a HR of 2.52  for overall survival, a HR of 2.22  for disease-specific survival, and a HR of 1.97  for disease-free survival. The worse prognosis of T2N0M0-Immune cases was confirmed in multivariable analyses with a HR of 2.87  for overall survival; HR of 2.41  for disease-specific survival; and HR of 1.97  for disease-free survival. Furthermore, Kaplan–Meier survival curves (p = 0.003) in univariable analysis and a HR of 2.79  in the multivariable analysis. With regard to regional recurrence, however, there was no significant difference (p > 0.05) between T1N0M0-Immune and T2N0M0-Immune.In the analysis of the same cohort  staging system that can be routinely implemented in the clinical setting to identify aggressive cases of early OTSCC.Tumor cells interact with the surrounding immune microenvironment and this interaction is a major player during tumorigenesis and it has a valuable clinical significance . EvasionTILs have been studied in different subsites of head and neck cancer . The subA few limitations of this study should be mentioned. It is a retrospective study and examined the proposed TNM-Immune staging system in only one subsite of the oral cavity . The relationship between TNM-Immune and some variables including neck dissection, surgical margins, or adjuvant treatments was not analyzed in this study due to lack of data in many cases. In addition, a digital scoring of the immune response was not considered. These limitations need to be addressed in future studies.In early-stage OTSCC, there is a lack of evidence on the TNM-Immune classification. Our study reports for the first time the significance of including an immune parameter as a part of the staging system of early-stage OTSCC. TILs score, as a valuable indicator of the immune response, can routinely be included in the clinical practice and seems worth implementing in the staging system. Classification of OTSCC tumors staged as T1-T2N0M0 can be refined by TNM-Immune staging to recognize cases at a high risk of a worse outcome. It is necessary to initiate prospective studies, preferably multi-institutional, as a further step toward the introduction of TNM-Immune staging as part of routine prognostication and therapeutical decision-making in early OTSCC.


Methamphetamine (METH) abuse causes irreversible damage to the central nervous system and leads to psychiatric symptoms including depression. Notably, METH-induced hyperthermia is a crucial factor in the development of these symptoms, as it aggravates METH-induced neurotoxicity. However, the role of hyperthermia in METH-induced depression-like behaviors needs to be clarified. In the present study, we treated mice with different doses of METH under normal (NAT) or high ambient temperatures (HAT). We found that HAT promoted hyperthermia after METH treatment and played a key role in METH-induced depression-like behaviors in mice. Intriguingly, chronic METH exposure  or administration of an escalating-dose  of METH under NAT failed to induce depression-like behaviors. However, HAT aggravated METH-induced damage of hippocampal synaptic plasticity, reaction to oxidative stress, and neuroinflammation. Molecular hydrogen acts as an antioxidant and anti-inflammatory agent and has been shown to have preventive and therapeutic applicability in a wide range of diseases. Coral calcium hydride (CCH) is a newly identified hydrogen-rich powder which produces hydrogen gas gradually when exposed to water. Herein, we found that CCH pretreatment significantly attenuated METH-induced hyperthermia, and administration of CCH after METH exposure also inhibited METH-induced depression-like behaviors and reduced the hippocampal synaptic plasticity damage. Moreover, CCH effectively reduced the activity of lactate dehydrogenase and decreased malondialdehyde, TNF-α and IL-6 generation in hippocampus. These results suggest that CCH is an efficient hydrogen-rich agent, which has a potential therapeutic applicability in the treatment of METH abusers. Long-teStudies have shown that dopamine oxidative stress, excitotoxicity, and neuroinflammation are the most important mechanisms in METH-induced neurotoxicity . Additioad libitum hydrogen-rich water (HRW) consumption significantly inhibited METH-induced spatial memory impairment in the Barnes and Morris water maze tests , forced swimming test (FST), and locomotion test (LMT). In addition, the effect of CCH on METH-induced hyperthermia and depression-like behaviors was also explored. As the damage of hippocampal neurons plays a key role in plasticity regulation of synapses and a critical role in the mechanism of depression, Golgi staining in hippocampus was conducted, and the index of oxidative stress and neuroinflammation were also measured by the detection of lactate dehydrogenase (LDH), malondialdehyde (MDA), IL-6 and TNF-α levels.In this study, animals were treated with METH in different ambient temperatures to investigate the role of hyperthermia in METH-induced depression-like behaviors ad libitum and were kept in a climate controlled environment, at a consistent temperature (22 ± 1°C), humidity (approximately 60%), and a 12-h light/dark cycle (lights off at 7:00 a.m.). All experimental procedures were approved by the Local Animal Use Committee of Hebei Medical University and performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.Three hundred and ninety C57BL/6 mice, initially weighing 20–22 g (8 weeks old) were ordered from Beijing Vital River Laboratory Animal Technology Co., Ltd., China. All the mice were provided with food and water DL-METH was provided by Beijing Municipal Public Security Bureau, China. CCH was provided by Shanghai Quanren Biological Technology Co., Ltd. . The stock solution of METH (1 g/mL) was dissolved in 0.9% sterile saline, and CCH was suspended in pure water before use. The concentration of METH was adjusted to an appropriate injection volume of 10 mL/kg of body weight in each experiment.The procedure of behavioral testing was consistent with previous studies . Four brFour transparent resin cylinders with a diameter of 10 cm and a height of 23 cm were filled with 15 cm of 23–25°C warm water for performing the FST. Each mouse was placed in a cylinder and videotaped for 6 min to record the immobility time. The immobility in the last 5 min was measured and analyzed using Noldus Video Tracking Software. Immobility was defined as the absence of limb or body movements, except for what is necessary to keep the body from sinking. During the test, mice were separated from each other to prevent visual and acoustic interplay. The used water was replaced with fresh water after each test.The mice were placed in a brightly lit 40 cm × 40 cm white plexiglass arenas. The movement and location of the mice were recorded. The total distance traveled within the arena was recorded for a single 5 min session, which was used to measure the motor ability of the mice. The arena was cleaned with 75% alcohol between each test.via intraperitoneal (i.p.) injections with a 2-h interval in between each injection for 1 or 3 days. Two hours after the last METH injection, animals were placed back to their home cages at normal ambient temperature (22°C). The core body temperature of each mouse was recorded at 1-h after the first and second METH injection. Also, the core body temperature and body weight were measured at 24-h after the first METH injection. The depression-like behaviors were tested 7 and 14 days after METH exposure. All mice were tested in the following order: LMT, TST, and FST.The drug exposure regimen and dose described in previous studies were used in the present study. As shown in Subsequently, the effect of different drug exposure regimens and doses on METH-induced depression-like behaviors was investigated . Two batMice were pre-treated with CCH  resuspended in 0.2 mL pure water 1-h before METH exposure  at 28°C ambient temperature. The core body temperature was determined every 20 min for 2-h. To explore the effect of CCH on METH-induced depression-like behaviors, mice were treated with METH (10 mg/kg × 4) for 3 days and then administered with pure water  or CCH  twice per day. The depression-like behaviors were tested 7 and 14 days after METH exposure by TST, FST, and LMT.Mice were treated with METH (10 mg/kg × 4) for 3 days and then administered with pure water  or CCH  twice per day. Golgi staining was performed to examine the effect of METH exposure and CCH administration on the hippocampal synaptic plasticity. Hippocampal tissues were dissected to detect the level of oxidative stress products (MDA and LDH) and inflammatory cytokines (TNF-α and IL-6) by commercial assay kits.Golgi staining was used to detect the changes of dendritic spines in hippocampal neurons. The commercial Golgi staining kit made by Genmed Medicine Technology Co., Ltd.  was used. The mice were anesthetized with isoflurane and perfused with 1 × PBS solution and 4% paraformaldehyde. The brains of these mice were harvested and washed with pure water, then placed in a soak solution for 2 weeks in the dark and subsequently transferred to 30% sucrose solution for 48-h. Sagittal sections (80 ∼ 100 μm thick) were stained using the oscillating sectioning technique. One out of every five sections were selected, and a total of three sections were selected from each mouse. After Golgi staining, the changes of dendritic spines were observed under a microscope, and the number of dendritic spines was calculated by two independent observers.The levels of TNF-α and IL-6 in hippocampal tissues were detected by enzyme-linked immunosorbent assay (ELISA). The experiment was conducted according to the product instruction . The levels of MDA and LDH, which are the markers of lipid peroxidation, were determined using a thiobarbituric acid (TBA) assay kit and 2,4-dinitrophenylhydrazine colorimetric assay kit  in accordance with the manufacturer’s protocol. After completing the drug treatments, hippocampal tissues of six mice in each group were collected and stored at −80°C until analysis. When performing the experiments, the tissues were homogenized in PBS buffer and centrifuged to collect the supernatant. The total protein content was tested using a Bicinchoninic Acid (BCA) protein assay kit . The levels of TNF-α, IL-6, MDA and LDH was measured in nanomole per microgram (nmol/mg) of protein.post hoc test was performed to assess the differences between groups. Unpaired two-tailed Student’s t-test was used to compare two independent groups. The threshold for statistical significance was set at P < 0.05 .Data are presented as means ± standard error of mean (SEM). Analysis of variance (ANOVA) including one-way, two-way, and three-way ANOVA and mixed ANOVA (repeated-measures design) were used for the statistical analyses. Bonferroni’s A total of 390 C57BL/6 mice were initially purchased. With regard to accidental death after METH treatment, especially under HAT, the number of mice in each group was inconsistent in experimental design. In total, 24 mice died during METH treatments, 6 mice died because the failure of intragastric injection, and 5 mice were excluded due to poor general state and low activity in locomotion test. Thus, a total of 355 experimental animals were included in the data analysis.F1,54 = 57.29, P < 0.001) and ambient temperature , and significant interaction of METH treatment with ambient temperature . In addition, the two-way ANOVA revealed significant main effects on body weight loss for METH treatment , ambient temperature , and interaction of METH treatment and ambient temperature . Post hoc comparisons indicated that the body weight loss was much more serious when METH was given under HAT (P < 0.001), compared to NAT.Mice were treated with four doses of 10 mg/kg METH with 2-h interval in between doses at normal (22°C) and high (28°C) ambient temperatures , and theP = 0.782; T2: P = 0.390) and FST . However, METH treatment given with HAT induced depression-like behaviors both in TST (P < 0.001) and FST (P < 0.001) at 7 days after METH exposure (T1) but recovered in T2 tests . All the LMT results showed no significant differences between groups in T1 and T2 tests .To investigate the potential effects of hyperthermia on depression-like behaviors, mice were treated with METH (10 mg/kg × 4 injections) at NAT (22°C) and HAT (28°C), and the behavioral tests including LMT, TST, and FST were performed 7 (T1) and 14 (T2) days later. As shown in P = 0.568; FST: P = 0.611) and T2  tests when METH treatment was performed under NAT. However, METH treatment given with HAT induced long-lasting depression-like behaviors at least for 14 days. The results of FST and TST test revealed significant differences between saline and METH group in T1  and T2  tests. Moreover, METH treatments under NAT and HAT did not affect the locomotion of animals in T1 and T2 procedures .Next, we increased the doses of METH treatments to 10 mg/kg × 12 injections (in 3 days). The results of behavioral tests also showed no difference between groups in T1 (TST: P < 0.001).Interestingly, we involved different METH exposure regimens and doses under NAT with the aim to induce depression-like behaviors. As shown in F3,35 = 4.007, P = 0.015) and time , and interaction of drug treatment and time . Moreover, the one-way ANOVA revealed significant difference in body temperature at 60 min after METH treatment between groups . Post hoc comparisons indicated a rise in body temperature in METH-treated mice (P < 0.001), and an inhibitory effect of CCH pretreatment at doses of 200 mg/kg (P = 0.002), but not 100 mg/kg (P = 0.801) on METH-induced increase of body temperature , and received METH injection  1-h later under HAT (28°C) . The corperature .F3,28 = 18.74, P < 0.001) and FST . Post hoc comparisons indicated significant therapeutic effect of 100 mg/kg  and 200 mg/kg  CCH on METH-induced depression-like behaviors. Furthermore, there were no differences noted on the total distance in LMT between groups .To examine the therapeutic effect of CCH on METH-induced depression, CCH  treatments were given to mice for 7 days (once per day) after METH injections  . As showP = 0.027, F1,8 = 176.20, P < 0.001) and ambient temperature , but no interaction of METH treatment and ambient temperature . Furthermore, METH induced oxidative stress and neuroinflammation in a time-dependent manner. The one-way ANOVA indicated significant main effects on MDA levels , LDH , TNF-α , and IL-6  for METH treatment. HAT aggravated METH-induced abnormal changes in MDA, TNF-α and IL-6 level . Post hoc comparisons indicated that CCH administration effectively reversed METH-induced hippocampal synaptic plasticity damage . Similar results were revealed in MDA , LDH , TNF-α , and IL-6  content measurement , LDH , TNF-α , and IL-6 . Therefore, it can be stated conclusively that CCH administration significantly reversed hippocampal synaptic plasticity damage and alleviated oxidative stress and neuroinflammation induced by METH exposure.Golgi staining showed that METH elicited hippocampal synaptic plasticity damage in mice, and this change was more severe when METH was given under HAT  aggravates METH-induced hyperthermia and plays a key role in METH-induced depression-like behaviors. Secondly, we further clarified the effect of CCH, on METH-induced hyperthermia and behavioral abnormality. Treatment with CCH significantly inhibited METH-induced hippocampal synaptic plasticity damage and attenuated the rise in oxidative stress products and inflammatory cytokines in the hippocampus. Therefore, our study suggested that CCH used as an efficient hydrogen-rich agent is a novel and effective treatment of METH-induced psychiatric disorders.It is known that frequent use of psychostimulants causes elevated behavioral and cognitive activity, and can also lead to severe psychiatric symptoms . The symad libitum hydrogen-rich water consumption, and inhalation of HG were common methods to deliver molecular hydrogen to animals in experimental research. However, due to the low solubility of hydrogen in water, it is not easy to realize high concentration and long-term accumulation by hydrogen-rich water consumption or hydrogen-rich saline injection to animal model in a particular point of time. Therefore, we only evaluated the inhibitory effect of hydrogen on low dose METH-induced behavioral abnormality and neurotoxicity. Recent studies demonstrated that the hydrogen level released by CCH administration was more reliable and robust than hydrogen-rich water in vitro and in vivo. Moreover, 2+ levels after CCH treatment (Molecular hydrogen, as a selective antioxidant, was first reported by reatment . TherefoAlthough depressive symptoms and METH withdrawal have common neurobiological symptoms, our search for possible mechanisms of METH-induced depression-like behavior was focused on the METH-induced neurotoxicity and the involvement of hippocampal synaptic plasticity damage . Recent However, there have two limitations of the present study. Firstly, although the present data revealed the inhibitory effect of CCH on METH-induced severe hyperthermia, whether it affect the hypothalamus or generate peripheral effect thereby causing temperature change in METH-treated mice should be explored. Moreover, since the evidence of CCH toxicity in humans is limited, the possible toxicity should be carefully investigated in future studies.via the regulation of hippocampal synaptic plasticity damage mediated by oxidative stress and neuroinflammation. In summary, based on our present study, it can be concluded that CCH acts as a protective antioxidant treatment and may have potential application in reducing the risk of psychiatric symptoms in METH abusers.In the present study, we revealed that hyperthermia plays a key role in METH-induced depression-like behaviors. In addition, it was also proved that molecular hydrogen released by CCH effectively ameliorated METH-induced hyperthermia and depression-like behaviors, and this function was possibly elaborated The original contributions presented in the study are included in the article/The animal study was reviewed and approved by Hebei Medical University.DW and CM designed the experimental plans. XW, BT, and RH performed the behavioral part of the study. CH and ZZ did the molecular biological experiment in text. LZ and BX analyzed data. ZN and BC wrote and revised the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Doxorubicin (DOX) is an anthracycline anticancer drug, which is often associated with drug resistance and cytotoxicity. More unfortunately, the biological barrier in the human environment can weaken the efficacy of DOX, such as the blood-brain barrier (BBB). This work attempts to make efforts to solve this problem. We used polyethylene glycol distearoylphosphatidylethanolamine (PEG-DSPE) as a nanocarrier and DOX as a model drug to construct a composite nanodrug (TF-PEG-DSPE/DOX NPs) by coupling transferrin (TF). The results of glioma experiments show that the nanodrug can effectively penetrate BBB to achieve an antitumor effect. The rapid development of medical technology provides effective solutions for many clinical diseases. However, a radical cure for cancer has not been achieved in the current medical community. As a kind of malignant tumor that originated from the brain neuroectoderm, glioma has strong aggressiveness and high mobility. The five-year survival rate of patients is less than 10%, and the difficulty of treatment is self-evident .At present, surgical resection is the first choice for the treatment of glioma at home and abroad, supplemented by radiotherapy and chemotherapy –8. With DOX is a common antibiotic tumor drug, and its structure is shown in Recently, some researchers have been inspired by the specific binding process between transferrin (TF) and its receptor. Overexpression of TF receptors in glioma cells greatly increases the ability of tumor drugs carrying TF to penetrate BBB –28. Therin vitro and in vivo experiments of TF-PEG-DSPE/DOX NPs were carried out orderly  were prepared. In addition, in order to investigate the antitumor effect of the nanoparticles, the  orderly .44H87N2O10P) was purchased from Shenzhen Meiluo Technology Co., Ltd. . Doxorubicin  was purchased from Hubei Yongkuo Technology Co., Ltd. . Dimethyl sulfoxide  was purchased from Shanxi Jinyang Pharmaceutical Excipients Co., Ltd. . Triethylamine  was purchased from Jinan Yeqing Biotechnology Co., Ltd. . Transferrin (TF) was purchased from Shanghai Qiyuan Biotechnology Co., Ltd. . Brain endothelioma adherent cell line bEnd.3 and glioma cell line U87 were purchased from Shanghai Binsui Biotechnology Co., Ltd. . Thiazolyl blue  was purchased from Wuhan Kanos Technology Co., Ltd. . Phosphate-buffered saline (PBS) was purchased from Shanghai Jizhi Biochemical Technology Co., Ltd. . Chloral hydrate  was purchased from Shanghai Xinyu Biotechnology Co., Ltd. .Polyethylene glycol phospholipid .Weigh 1.0 g of PEG-DSPE and 250 mg of DOX and dissolve in 10.0 mL of DMSO solution. Add 0.10 mL of TEA solvent dropwise to the reaction system, then place at a 40°C water bath constant temperature shaker and wait for the reaction for one day to prepare a PEG-DSPE/DOX NP solution. Finally, TF was used to specifically bind the DSPE group of PEG-DSPE/DOX NPs by the TF thiolation method to obtain TF-PEG-DSPE/DOX NPs.4 cells/well and placed in the lower chamber containing 10% fetus. A fresh medium of bovine serum is cultivated. Closely observe the compactness of the cell monolayer. When the transmembrane resistance value of the monolayer cells is greater than 200 Ω/cm2, change the medium to a cell culture medium without fetal calf serum, and DOX, PEG-DSPE/DOX NPs, and TF-PEG-DSPE/DOX NPs were added to the upper chamber; 200 μL of culture medium was removed every 6.0 h, and the same amount of fresh culture medium was replaced in the lower chamber. Use a microplate reader to detect the fluorescence intensity at 630 nm.The performance of single DOX drugs, PEG-DSPE/DOX NPs, and TF-PEG-DSPE/DOX NPs in penetrating the blood-brain barrier was compared. Briefly, the logarithmic growth phase mouse brain endothelioma adherent cell line bEnd.3 was inoculated into the upper chamber of a 24-well plate Transwell with a concentration of 5.0 × 104 cells/well. After one day of culture, add 150 μL of DOX, PEG-DSPE/DOX NP, and TF-PEG-DSPE/DOX NP solutions . After the cells are cocultured with the drug for one day, add 25 μL of MTT solution to the 96-well plate and incubate for 4.0 h. After removing the supernatant, add DMSO solution dropwise to the culture plate to stop the entire reaction system. Record the absorbance of the reaction products of each experimental group at 490 nm to obtain the inhibitory rate of the drug on U87 cells.The MTT method was used to evaluate the inhibitory effect of TF-PEG-DSPE/DOX NPs on the glioma cell line U87. Briefly, the diluted U87 cell suspension was seeded in a 98-well plate at a concentration of 1.0 × 10in vitro. Briefly, adjust the pH of the PBS = 7.4, and dissolve 10.0 mg of TF-PEG-DSPE/DOX NPs in the PBS. Place the dialysis bag in an incubator at a constant temperature of 37°C for continuous shaking, and collect at preset time points  2.0 mL of dialysis external fluid, and use PBS to refill in time. The sample was filtered, and the drug release was determined by high-performance liquid chromatography.Use the dialysis method to explore the cumulative release of TF-PEG-DSPE/DOX NPs μL of U87 cells in the logarithmic growth phase (concentration of 1.5 × 106 cells/μL) into the mouse brain. After 10 min of observation, slowly take out the microsyringe. The incision was sutured after washing and disinfection, and 1.5 × 105 U penicillin was injected daily for preinfection within 7 d after surgery.In order to evaluate the targeted antitumor effect of TF-PEG-DSPE/DOX NPs, 84 BALB/c nude mice aged 6–8 weeks were selected as experimental subjects in this study. All mice were fasted and deprived of water within 12 h before the start of the operation and anesthetized by intraperitoneal injection of TCA (2.5 mL/kg). Fix the mouse on the stereotaxic device, and drill a 2.0 mm diameter hole on the midline of the forehead after skin preparation. Keep the microsyringe with a 5.0 mm needle, and then slowly inject 2.0 After the glioma model was successfully induced, they were randomly divided into the DOX group, PEG-DSPE/DOX NP group, and TF-PEG-DSPE/DOX NP group. The model mice of each group were treated differently by tail vein injection of drugs, and the corresponding drugs were injected at 4.0 mg/kg daily.The mice in each experimental group were randomly divided into two subgroups according to their body weight. The mice in the first subgroup were sacrificed at 0.5 h after the administration. The tumor tissues, heart, brain, liver, and kidney organs were taken, and the distribution of TF-PEG-DSPE/DOX NPs in the mice was recorded.After regularly treating the mice in the second subgroup, the survival time and weight of the mice in the DOX group, PEG-DSPE/DOX NP group, and TF-PEG-DSPE/DOX NP group were recorded to evaluate the antitumor activity of the drug.The transmission electron microscopy (TEM) image of TF-PEG-DSPE/DOX NPs is shown in in vitro model are shown in P > 0.05), while the penetration effect of TF-PEG-DSPE/DOX NPs is significantly better than that of DOX and PEG-DSPE/DOX NPs (P < 0.05). At 12, 18, and 24 h, the penetration rates of PEG-DSPE/DOX NPs and TF-PEG-DSPE/DOX NPs were higher than those of DOX (P < 0.05). Among them, the penetration efficiency of TF-PEG-DSPE/DOX NPs at the 24 h node is as high as 7.79 ± 0.37%. Therefore, the TF-PEG-DSPE/DOX NPs prepared in this experiment have great potential in the treatment of glioma.The test results of TF-PEG-DSPE/DOX NPs crossing the BBB P < 0.05), and when the drug concentration is 50 μM, the cell inhibition rate is about 88.23 ± 2.55%. It can be seen that TF-PEG-DSPE/DOX NPs have good antitumor effects.The experimentally observed inhibition rate of TF-PEG-DSPE/DOX NPs on U87 glioma cells is shown in in vitro release curves of different drugs under pH = 7.4 are shown in in vitro release rate of TF-PEG-DSPE/DOX NPs reached the maximum. The results show that the experimentally prepared TF-PEG-DSPE/DOX NPs have excellent sustained release properties and can effectively solve the problem of low DOX bioavailability.The Through experiments in mice, this work obtained the tissue distribution of different drugs in mice, and the results are shown in The survival record of the model mice after treatment is shown in The degree of malignancy of glioma is well known. At present, although many medical techniques have been applied to the disease management of glioma, the results of practice are often unsatisfactory. For example, DOX is an antibiotic drug, which often appears in cancer treatment programs. However, after a long period of research, it is found that the existence of BBB restricts the antitumor effect of drugs, which points out the research direction for medical personnel .in vitro drug release experiments show that the sustained release effect of TF-PEG-DSPE/DOX NPs meets expectations. In addition, TF-PEG-DSPE/DOX NPs performed well in penetrating BBB model experiments. Of course, research on the role of TF-PEG-DSPE/DOX NPs in glioma animal models is also being carried out in subsequent experiments. The results show that TF-PEG-DSPE/DOX NPs can effectively prolong the life cycle of glioma mice. In addition, the retention concentration in the kidney tissue of the body decreases while the concentration in the brain tissue increases, indicating the drug's effects on the kidney tissue; the burden is small.Considering the difficulties at this stage, this project has synthesized a nanodrug (TF-PEG-DSPE/DOX NPs) combined with multiple biomolecules. In the initial stage of the experiment, the morphology of TF-PEG-DSPE/DOX NPs was characterized, and it was found that the nanoparticles were uniform in size and stable in physical and chemical properties. Secondly, in vivo and in vitro experiments have confirmed the role of TF-PEG-DSPE/DOX NPs in glioma, which provides data for clinicians to optimize treatment plans.In summary, In vitro experiments prove that the experimentally synthesized TF-PEG-DSPE/DOX NPs can effectively penetrate BBB and have excellent drug sustained release properties. In addition, in vivo animal experiments based on BALB/c nude mice have verified the targeted antitumor effect of TF-PEG-DSPE/DOX NPs in the body. Therefore, TF-PEG-DSPE/DOX NPs nanoparticles are just around the corner in the clinical treatment of glioma.The low bioavailability of DOX is the main problem that plagues surgeons. This work has made efforts in this regard.